1. From basic Concepts to Advanced applications Molecular Evolution & Phylogeny By Ofir Cohen The Bioinformatics Unit G.S. Wise Faculty of Life Science Tel Aviv University, Israel Feb 2011 http://ibis.tau.ac.il/twiki/bin/view/Bioinformatics/Phylogeny

2. 2 of 20 Part II: Tools

3. 3 of 20 Multiple sequence alignment (MSA) Several advanced MSA programs are available. Today we will use two: MAFFT – fastest and one of the most accurate PRANK – distinct from all other MSA programs because of its correct treatment of insertions/deletions Tools - Alignments

4. 4 of 20 MAFFT Web server (& download option): http://mafft.cbrc.jp/alignment/server/index.html http://mafft.cbrc.jp/alignment/server/index.html Efficiency-tuned variants  quick & dirty or slow but accurate Nucleic Acids Research, 2002, Vol. 30, No. 14 3059-3066 © 2002 Oxford University PressOxford University Press MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform Kazutaka Katoh, Kazuharu Misawa 1, Kei-ichi Kuma and Takashi Miyata * Tools - Alignments

5. 5 of 20 Choosing a MAFFT strategy quick & dirty slow but accurate Tools - Alignments

6. 6 of 20 MAFFT output Saving the output Choose a format: Clustal, Fasta, or click "Reformat" to convert to a selection of other formats Save page as a text file A colored view of the alignment Tools - Alignments

7. 7 of 20 PRANK Tools - Alignments

8. 8 of 20 Classical alignment errors for HIV env Tools - Alignments CLUSTALW PRANK

9. 9 of 20 PRANK Web server: http://www.ebi.ac.uk/goldman-srv/webPRANK/http://www.ebi.ac.uk/goldman-srv/webPRANK/ Tools - Alignments

10. 10 of 20 PRANK output If you need a different format – copy the results to the READSEQ sequence converter: http://www-bimas.cit.nih.gov/molbio/readseq/ http://www-bimas.cit.nih.gov/molbio/readseq/ Tools - Alignments

11. 11 of 20 Downloadable PRANK http://www.ebi.ac.uk/goldman-srv/prank/prank/  PRANK: A command-line program interface  PRANKSTER: A program with graphical user interface Tools - Alignments

12. 12 1.Download the sequence files from the web-site http://ibis.tau.ac.il/twiki/bin/view/Bioinformatics/Phylogeny Open "fahA.fas" in Notepad/Browser – these are 65 protein sequences in FASTA format. http://ibis.tau.ac.il/twiki/bin/view/Bioinformatics/Phylogeny 2.Run PRANKSTER, http://www.ebi.ac.uk/goldman-srv/prank/src/prankster/windows/ open the "fahA.fas" file, and run "Alignment"  "Make alignment" http://www.ebi.ac.uk/goldman-srv/prank/src/prankster/windows/ 3.While you wait… Copy the sequences into the MAFFT web server http://mafft.cbrc.jp/alignment/server/index.html run the "auto" strategy. (which strategy did MAFFT choose for you?) Click "Reformat", choose "phylip|phylip4", and save as "fahA.mafft.phylip" http://mafft.cbrc.jp/alignment/server/index.html 4.…When PRANKSTER finishes click File  Save, and save the MSA in Phylip format by the name "fahA.prank.phylip“ 5.If you have time, run PRANK web server http://www.ebi.ac.uk/goldman-srv/webPRANK/ http://www.ebi.ac.uk/goldman-srv/webPRANK/ 6. (1)

13. 13 of 20 Trees Reconstruction Methods

14. 14 of 20 Phylogeny reconstruction Different approaches (algorithms / programs): Distance based methods (e.g. neighbor-joining, as in ClustalW)  Fast but inaccurate Maximum parsimony (e.g. MEGA)MEGA Maximum likelihood methods (e.g. phyML, RAxML)  Accurate but slowerphyMLRAxML Bayesian methods (e.g. MrBayes)  Most accurate but very slowMrBayes ABCDEABCDE Guide tree A D C B E MSA Pairwise distance table Tools - Trees

15. 15 of 20 PhyML The most widely used maximum likelihood (ML) program Web server (& download) : http://www.atgc-montpellier.fr/phyml/http://www.atgc-montpellier.fr/phyml/ Tools - Trees

16. 16 1.Give "fahA.prank.phylip" or "fahA.mafft.phylip" as input to the phyML webserver (don't forget to choose "Amino-acids" and enter your email and name) (2)

17. 17 of 20 RAxML Web server: http://phylobench.vital-it.ch/raxml-bb/http://phylobench.vital-it.ch/raxml-bb/ Similar maximum likelihood (ML) methodology as phyML, but much faster  Faster results  Better results in same run-time Tools - Trees

18. 18 1.Give "fahA.prank.phylip" or "fahA.mafft.phylip" as input to the RAxML webserver (don't forget to tick "Protein sequences" and enter your email) Save the resulting tree file as: "fahA.prank.phylip.raxml" (3)

19. 19 of 20 FigTree: tree visualization and figure creation http://tree.bio.ed.ac.uk/software/figtree/ http://tree.bio.ed.ac.uk/software/figtree/ Manipulate a node Manipulate a clade Manipulate a taxon

20. 20 1.In case tree are not ready yet… download tree from website 2.Open "fahA.prank.phylip_phyml_tree.txt" in FigTree http://tree.bio.ed.ac.uk/software/figtree/ http://tree.bio.ed.ac.uk/software/figtree/ 3.Play around with the different options and make a pretty figure! 1.Find out how to color specific clades, as below 2.Try each of the three options under "Layout" 4.Export a figure in PDF format (File  Export Graphic…) (4)

21. 21 of 20 Final Questions… Thanks for your attention